Cardoso et al. BMC Evolutionary Biology (2015) 15:106 DOI 10.1186/s12862-015-0383-4

RESEARCH ARTICLE Open Access Phylogeography of the sand dune Mycetophylax simplex along the Brazilian Atlantic Forest coast: remarkably low mtDNA diversity and shallow population structure Danon Clemes Cardoso1,2,3*, Maykon Passos Cristiano3,4, Mara Garcia Tavares1, Christoph D. Schubart3 and Jürgen Heinze3*

Abstract Background: During past glacial periods, many species of forest-dwelling experienced range contractions. In contrast, species living outside such moist habitats appear to have reacted to Quaternary changes in different ways. The Atlantic Forest represents an excellent opportunity to test phylogeographic hypotheses, because it has a wide range of vegetation types, including unforested habitats covered predominantly by herbaceous and shrubby plants, which are strongly influenced by the harsh environment with strong wind and high insolation. Here, we investigated the distribution of genetic diversity in the endemic sand dune ant Mycetophylax simplex across its known range along the Brazilian coast, with the aim of contributing to the understanding of alternative phylogeographic patterns. We used partial sequences of the mitochondrial gene cytochrome oxidase I and nuclear gene wingless from 108 specimens and 51 specimens, respectively, to assess the phylogeography and demographic history of this species. To achieve this we performed different methods of phylogenetic and standard population genetic analyses. Results: The observed genetic diversity distribution and historical demographic profile suggests that the history of M. simplex does not match the scenario suggested for other Atlantic Forest species. Instead, it underwent demographic changes and range expansions during glacial periods. Our results show that M. simplex presents a shallow phylogeographic structure with isolation by distance among the studied populations, living in an almost panmictic population. Our coalescence approach indicates that the species maintained a stable population size until roughly 75,000 years ago, when it underwent a gradual demographic expansion that were coincident with the low sea-level during the Quaternary. Such demographic events were likely triggered by the expansion of the shorelines during the lowering of the sea level. Conclusions: Our data suggest that over evolutionary time M. simplex did not undergo dramatic range fragmentation, but rather it likely persisted in largely interconnected populations. Furthermore, we add an important framework about how both glacial and interglacial events could positively affect the distribution and diversification of species. The growing number of contrasting phylogeographic patterns within and among species and regions have shown that Quaternary events influenced the distribution of species in more ways than first supposed. Keywords: Brazilian Atlantic Forest, Mycetophylax simplex, Formicidae, Neotropical region, Gene flow, Genetic structure, Sand dunes, Restinga, Sea-level changes

* Correspondence: [email protected]; Juergen. [email protected] 1Present address: Departamento de Genética, Universidade Federal do Paraná, Setor de Ciências Biológicas, Rua Francisco H. dos Santos, s/n°, Jardim das Américas, Curitiba, Paraná 81530-000, Brazil 3Zoology/Evolutionary Biology, Universität Regensburg, Universitätstrasse 31, D-93040 Regensburg, Germany Full list of author information is available at the end of the article

© 2015 Cardoso et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Cardoso et al. BMC Evolutionary Biology (2015) 15:106 Page 2 of 13

Background with the cyclical shrinking or expanding of forests. Alter- Climatic fluctuations during the late Quaternary associ- natively, their distribution may have remained largely ated with the Last Glacial Maximum have had a strong unchanged during these historical events. While several impact on the current distribution of many and species associated with humid forest environments have plant species worldwide. Climate change not only af- been used to infer the evolutionary processes that oc- fected the landscape of continental areas but also the sea curred during the Quaternary in the Atlantic Forest, only level, which in turn shaped coastal landscapes by form- a few studies have addressed organisms – exclusively ing land bridges, islands, sand coastal plains, as well as plants – associated with dry environments (see [17]). connecting and separating areas [1, 2]. All these climate- Mycetophylax simplex is a small fungus-growing ant linked processes may have influenced the evolutionary (Formicidae: ) endemic to sand dune envi- history of the species, especially those inhabiting coastal ronments along the Brazilian coast, occurring from the areas [3, 4]. southern São Paulo State to Rio Grande do Sul [22, 23]. Pollen data, fossil records, and paleoclimatic data indi- Although this ant exhibits a wide distribution, it is re- cate that numerous taxa in the Northern hemisphere stricted to specialized habitats (sand dunes), making it a were restricted during the period of glaciation to south- good model organism to test phylogeographic scenarios ern or eastern refugia [5], and phylogeographic studies for open and dry coastal environments. Thus, the aim of show how expansion from these sites has molded their this study is to evaluate the genetic relationship among distribution today [6–9]. In contrast, less is known about populations of M. simplex across its whole distribution these phenomena in the Southern hemisphere, where and to infer how the Quaternary oscillations affected the glacial refugia hypothesis only recently has been for- genetic diversity and structure of its populations. Based mally evaluated [10, 11]. It is presently the most frequently on DNA sequences of the mitochondrial gene cyto- suggested mechanism for the current distribution of spe- chrome oxidase subunit I (COI) and the nuclear gene cies diversity in the Brazilian Atlantic Forest (AF), the sec- wingless, we aimed to test if (i) M. simplex underwent a ond largest Neotropical forest after the Amazon rainforest. persistent range and population size during the climatic The AF extends more than 3300 km along the eastern oscillations of the Quaternary and if and how (ii) its coast of Brazil. It has received worldwide attention be- demographic history was affected by the cyclic changes cause of its high and high level of endemism in the sea level. and was determined as one of the 25 world biodiversity hotspots for conservation priorities [10, 12]. The AF pre- Methods sents a wide range of vegetation types with conspicuous Sampling and laboratory procedures changes across landscapes, which include open habitats A total of 108 colonies of M. simplex, spanning its whole covered predominantly by herbaceous and shrubby plants, distribution area, were collected from January to September which develop on marine deposits. Despite an increasing 2011 (Fig. 1). The geographical references and sample size number of phylogeographic studies, the knowledge about of all samples are given in Table 1. The specimens were the evolutionary history of the AF is still limited and con- preserved in 100 % ethanol until DNA extraction. Whole troversial [10, 13–19]. The refugia hypothesis has gained genomic DNA was extracted from one individual per support by a growing number of phylogeographic studies colony. Genomic DNA was isolated using proteinase K di- that attempt to explain the high diversity in the AF [13, gestion followed by a standard CTAB protocol [24]. Frag- 15, 16]. Basically, it states that during glaciation, when the ments of the mitochondrial gene cytochrome oxidase climate was drier in the Southern hemisphere, forests subunit I (COI) and the nuclear gene wingless, both contracted and persisted only in moister areas, which be- known to be useful in intra-specific studies in (e.g. came refugia for humidity-dependent species. By vicariant [25]), were amplified by PCR using the previously pub- processes, these refugia promoted speciation and therefore lished primers LCO1490 [26] and Ben [27] for COI and today harbor a higher genetic diversity and endemism Wg578F [28] and Wg1032R [29] for wingless.DNA- than areas that did not serve as refugia [1]. amplification was conducted in reactions with 25 μLfinal The retreat of forests and the changed global climate volume containing: MgCl2 (2.5 mM), buffer (10x; Pro- conditions may have allowed the expansion of drought- mega), dNTPs (1 mM each), two primers (0.48 μMeach), tolerant biomes [20, 21]. Species adapted to dry and Taq polymerase (2 U of GoTaq® Flexi DNA Polymerase) open habitats in southern and eastern South America, and 1 μL of template DNA. PCR cycling conditions were including the Atlantic coast, may have expanded during as follows: initial denaturation for 2 min at 94 °C, then the drier periods throughout climatic oscillations. Com- 35 cycles with 94 °C for 1 min denaturation, 50 °C (COI) pared to species restricted to refugia, such species may or 55 °C (wingless) for 1 min annealing, 72 °C for 2 min have experienced recurrent shifts in their distribution, extension, and a final extension at 72 °C for 7 min. PCR with populations mixing or separating from each other products were purified and sequenced by Macrogen Inc. Cardoso et al. BMC Evolutionary Biology (2015) 15:106 Page 3 of 13

Fig. 1 Map showing the 27 sampled localities throughout the distribution of M simplex on the southern and southeast Atlantic coast of Brazil. Each color square represents one population (for details see table 1). The red highlighted area on the southeast coast is the gap in the distribution of M. simplex where we did not find the species besides our sampling effort, and the dashed line represents the limits of the sea–level during the last glacial maximum (approximately 21 Mya). The map was produced using a GIS program with free layers available at IBGE website (www.mapas. ibge.gov.br)

(www.macrogen.com) in both directions using the same analysis was conducted only with the mitochondrial gene primers as for amplification in an ABI PRISM 3700 se- COI. The examination of additional nuclear genes (long- quencing system. Singletons were confirmed by sequen- wave rhodopsin (GenBank accession numbers: KC964627- cing a second individual from the same colony when KC964632) and abdominal A (data not show)) also failed available. to reveal intraspecific variability (for primers see [28]). Sequence variation was analyzed with the software Analysis MEGA 5.0, and diversity parameters, including nucleo- The chromatograms of each gene were evaluated and tide diversity (π) and haplotype diversity (h), were com- edited separately using the program Consed [30]. After- puted with DNASP 5 [32] for each population and for wards, they were analyzed by translation into amino the complete dataset. To ensure reliable estimates of acids using the program MEGA 5.0 [31] in order to regional differentiation and diversity and to allow ap- check for indels and premature stop codons. Since wing- propriate statistical analysis, geographically and eco- less did not show intraspecific variation throughout the logically close locations (neighboring beaches and/or sampled populations (51 specimens from same populations beaches inside large rivers basins) were pooled in order were analyzed (GenBank accession numbers: KP939178- to obtain ten populations with sample sizes with of at least KP939228), see Additional file 1: Appendix S1), subsequent eight colonies (in Table 1 sampling localities belonging to Cardoso et al. BMC Evolutionary Biology (2015) 15:106 Page 4 of 13

Table 1 Population details, geographical location of the population encompassing 27 sampled localities (S = latitude, W = longitude) throughout the range distribution of M. simplex and its haplotype distribution Population Locality Coordinate Haplotype (number) SW RS1 Chuí 33° 43’ 53° 21’ H5(3), H22(1), H26(1) Cassino 32° 13’ 52° 11’ H5(3), H14(1), H27(1) RS2 São José do Norte 32° 03’ 51° 59’ H5(3), H29(1) Mostardas 31° 07’ 50° 50’ H22(2), H25(1), H28(1), H30(1) RS3 Cidreira 30° 07’ 50° 11’ H2(2), H21(1), H22(1), H23(1) Curumim 29° 37’ 49° 56’ H2(1), H5(1), H19(1), H20(1) Torres 29° 21’ 49° 44’ H24(1) SC4 Bal. Arroio do Silva 29° 00’ 49° 26’ H1(1), H4(1) Bal. Gaivota 29° 11’ 49° 35’ H1(1), H4(1), H3(1), H7(1) Araranguá 28° 57’ 49° 22’ H1(2), H7(2), H10(1) SC5 Ilhas 28° 54’ 49° 19’ H1(1), H2(1), H30(1), H31(1) Bal. Rincão 28° 48’ 49° 12’ H1(1), H3(2), H4(1), H5(1) Laguna 28° 36’ 48° 50’ H3(1), H29(4) SC6 Itapirubá 28° 19’ 48° 42’ H3(1), H8(1) Garopaba 27° 59’ 48° 37’ H3(2), H9(1), H11(2) Pinheira 27° 50’ 48° 35’ H3(1), H11(3) SC7 Florianópolis – Moçambique 27° 29’ 48° 23’ H11(2) Florianópolis – Joaquina 27° 37’ 48° 27’ H1(1), H3(1), H11(2) Florianópolis – Pântano do Sul 27° 46’ 48° 31’ H3(1), H11(2), H13(1), H14(1) SC8 Gov. Celso Ramos 27° 19’ 48° 32’ H27(1), H28(1) Navegantes 26° 51’ 48° 38’ H11(4) São Francisco do Sul 26° 15’ 48° 31’ H1(1), H3(2), H12(1), H15(1) PR9 Guaratuba 25° 56’ 48° 34’ H1(1), H3(2), H11(2) Pontal do Paraná 25° 40’ 48° 27’ H3(1), H9(1), H11(1), H32(1) NE10 Ilha Comprida - Cananéia 25° 02’ 47° 53’ H3(2), H9(1), H11(2) Ilha Comprida - Iguape 24° 42’ 47° 28’ H3(2), H11(3) Cabo Frio 22° 54’ 42° 02’ H3(1), H6(1) the same population are shown in the same colors in using Tracer v1.4 [35] and a total of 25 % of the trees Fig. 1). were excluded as burn-out prior to producing a consen- We used Bayesian Inference with MrBayes 3.2 [33] to sus topology. Finally, this topology was presented using infer phylogenetic relationships among the haplotypes of the Figtree v. 1.3.1. software [36]. M. simplex and to assess the monophyletic status of this In order to assess the correlation between log- species. We selected the model of sequence evolution transformed genetic diversity and geographical distances that best fit our dataset using the Akaike’s information among the sampled populations we carried out a Mantel criterion (AIC) implemented in MrModeltest 2.3 [34] test using the program AIS (alleles in space) [37]. The and used Trachymyrmex jamaicensis (GenBank acces- genetic differentiation among M. simplex populations sion number: DQ353390) and Cyphomyrmex costatus was measured by means of F-statistics [38]. Pairwise (JQ617535 and JQ617502) as outgroups. The Bayesian comparisons of ΦST between populations were calculated analyses consisted of two independent runs of ten mil- using the program ARLEQUIN 3.5 [39], which allows esti- lion generations each, starting with a random tree and mating the degree of gene flow among populations of M. four Markov chains, sampled every 1000 generations. simplex. The convergence among runs was verified by the aver- We also carried out a spatial analysis of molecular age standard deviation of split frequencies that had to variance using the program SAMOVA 1.0 [40] to search reach < 0.01. An appropriated burn-in was determined for partitions of sampling sites that were genetically Cardoso et al. BMC Evolutionary Biology (2015) 15:106 Page 5 of 13

homogenous, but maximally differentiated from each were combined in TRACER 1.4.1 and checked for ad- other. Based on value K that needs to be optimized, this equate mixing of the MCMC chains by values of effective method uses simulated annealing procedures to seek the sample size (ESS). Finally, we used the mutation rate cal- best clustering option that can be defined between culated above with our intraspecific COI dataset in order groups of populations among group genetic variation co- to construct the timing and magnitude of changes in the efficients (FCT). Analyses were conducted five times to effective population size using the Bayesian skyline check consistency for different K values and based on method [42]. This model was used to estimate the effect- 1000 simulated annealing steps with K increasing from 2 ive population size through time, as the most recent to 9 as the number of considered populations in each common ancestor of M. simplex. Therefore, the BEAST analysis. We also carried out a second SAMOVA ana- software molecular clock was set to Uncorrelated Log- lysis for different K values increasing from 2 to 20 con- normal under a Relaxed Clock and analyses were per- sidering each sampling location separately (despite low formed using the SRD06 with distinct rates of sequence within location sample size) in order to obtain the re- evolution for each codon partition and a tree prior to co- sults without any a priori population pooling. Thus, we alescence (constant size) was employed. Results from could identify the clustering of samples that yielded the three independent runs with 50 million generations each largest, and most significant, FCT for a given K. (with the initial 10 % excluded as burn-out) were com- The genetic distances among M. simplex haplotypes bined and analyzed with Tracer 1.4.1. were reconstructed using two methods: the Median Join- ing network algorithm implemented in NETWORK 4.6 Results (http://www.fluxus-engineering.com) and the statistical Sequence variation, phylogenetic relationships and parsimony procedure for phylogenetic network estima- structure tion, with 95 % criterion for a parsimonious connection We aligned a total of 108 sequences of the gene COI. applied in TCS 1.21 [41]. After exclusion of sections at the beginning and end of Trends in the demographic history of populations of the sequences, which in several samples were not com- M. simplex were investigated using three different ap- plete, the analyzed fragment had a total length of 845 proaches. First, under the assumption of neutrality devi- nucleotides. The 32 unique haplotypes were widely ations in Tajima’s and Fu’s FS statistics, we tested for distributed across the range of Mycetophylax simplex past population expansions. A negative value in both (GenBank accession numbers: KJ842219-KJ8423260). statistic tests would reflect either purifying selection in a Of the 32 polymorphic sites found, 19 were singletons and population at mutation-drift equilibrium, or deviations 13 were parsimony-informative. The COI sequences had from mutation-drift equilibrium, resulting from popula- an A-T bias as in most other mitochondrial tion expansion events. Second, we observed the distribu- genes (see [44]) and a bias against guanidine (T: 36.2 %. C: tion of pairwise nucleotide differences among haplotypes 20.3 %, A: 31.9 % G: 11.6 %), and all nucleotide substitu- and tested the deviation from the expected mismatch dis- tions were located at the third codon position. tribution under sudden and spatial models by means of a The Bayesian phylogenetic analyses indicated that M. generalized least-squares method and Harpending’s h sta- simplex forms a well-supported monophyletic cluster tistics. Both analyses were carried out in Arlequin 3.5. relative to the outgroups (Additional file 2: Appendix S2), Third, we used a Bayesian skyline plot (BSP) approach and that all specimens studied throughout the distribution [42], implemented in BEAST 1.6.1 [43], with the aim of can be considered the same species. Overall, haplotype recovering changes in the effective population size (Ne) and nucleotide diversity was 0.865 ± 0.022 and 0.00346 ± over time. For this, we first estimated a mutation rate for 0.00019 (mean ± SD), respectively (Table 2). the Mycetophylax from the COI sequences and fos- The Median Joining haplotype network was in agree- sil calibration of the molecular phylogeny of the ant tribe ment with the statistical parsimony network. In the gene Attini (see [25]). Mutation rate estimates were performed genealogy among the haplotypes (considered a 95 % under an uncorrelated lognormal-relaxed clock model, threshold for the probability of a parsimonious connec- using the model of sequence evolution GTR + I + Γ with tion being achieved) all haplotypes were joined in a sin- three partitions (codons 1, 2, and 3) with a random start- gle network (Fig. 2). The network did not indicate ing tree following a Yule speciation process as a prior tree. divergent clades within the genealogy of M. simplex. Based on fossils corresponding to the Cyphomyrmex rimo- Two major haplotypes comprised 50 % of the individ- sus group, the root node was given a lognormal distribu- uals: 27.8 % of the individuals had haplotype H3, which tion with a mean of 1.6, standard deviation of 1.0 and appeared to be widespread across the species range, offset of 15, as described in Mehdiabadi et al. [25]. The re- whereas haplotype H11, present in 22.2 % of the individ- sults from three independent runs of 50 million genera- uals, was almost completely restricted to the north of tions sampled every 5,000 with a burn-in of 15 million the species range. H11 is separated from other haplotypes Cardoso et al. BMC Evolutionary Biology (2015) 15:106 Page 6 of 13

Table 2 Genetic diversity and neutrality tests for each population and with all populations of M. simplex together Populations Nucleotide diversity (π) (± S.D.) Haplotype diversity (h) (± S.D.) Tajima's D Fu's FS RS1 0.00189 (0.00090) 0.667 (0.163) −1.87333 (P=0.0083) −1.11562 (P=0.1609) RS2 0.00224 (0.00054) 0.889 (0.091) −0.6299 (P=0.2859) −2.32907 (P=0.0261) RS3 0.00276 (0.00055) 0.933 (0.077) −1.50661 (P=0.0632) −4.46904 (P=0.0025) SC4 0.00181 (0.00024) 0.818 (0.083) 0.43329 (P=0.6969) −1.02733 (P=0.1714) SC5 0.00268 (0.00036) 0.890 (0.060) −1.09063 (P=0.1463) −2.8844 (P=0.0302) SC6 0.00336 (0.00039) 0.709 (0.099) 1.52257 (P=0.9504) 1.62676 (P=0.8143) SC7 0.00387 (0.00064) 0.709 (0.137) 1.49895 (P=0.9408) 0.7727 (P=0.6626) SC8 0.00346 (0.00042) 0.873 (0.089) 1.00501 (P=0.8566) −1.68615 (P=0.1229) SC9 0.00368 (0.00059) 0.833 (0.098) 0.92757 (P=0.8263) 0.12678 (P=0.4978) NE10 0.00339 (0.00038) 0.697 (0.090) 1.68302 (P=0.9613) 1.85074 (P=0.8387) All populations 0.00346 (0.00019) 0.865 (0.022) −1.47062 (P=0.0422) −21.59803 (P=0.0001)

by two missing intermediate haplotypes (Fig. 2). Large on the coast. Considering the non-significant ΦST values, portions of the recovered haplotypes were singletons the haplotypes of M. simplex cluster in three major (Table 1), but no more than two nucleotide substitutions population groups: southern populations (RS1 and RS2), (mutation steps) separate neighboring haplotypes (Fig. 2). central-eastern populations (RS3, SC4, and SC5), and There was no clear association between genetic struc- northern populations (SC6 – NE10) (Table 3). ture and geography concerning haplotypes, since several In line with these results, the Mantel test showed that haplotypes were widespread within the range of M. sim- genetic and geographic distance are slightly correlated plex (Figs. 2 and 3). However, pairwise ΦST comparisons (r = 0.075, p = 0.0007), suggesting a weak degree of isola- between populations indicated a certain geographic tion by distance. The spatial analysis of molecular vari- structure among the haplotypes of M. simplex. Popula- ance implemented in SAMOVA was not able to identify tions from the southern edge of the distribution (RS1, possible breaks among populations. All parameters var- RS2) did not show significant genetic differentiation and ied only little (Fig. 4) and none of them suggested a bet- ΦST values were low (Table 3), suggesting the absence of ter explanation for the genetic structure of M. simplex. a considerable barrier to gene flow. The same homogen- FCT values differed slightly from each other (0.22 - 0.26) eity was observed in populations from the north (SC6 – and did not show any trend of increasing or decreasing NE10). However, gene flow between these northern and with K (potential subdivisions across populations). Be- southern populations appears to be restricted, which sides, the major proportion of variance was found within leads to the slight geographic structure observed in the populations, ranging from 76 % to 83 % (Fig. 4b). Like- distribution of M. simplex haplotypes likely due to wise, the second SAMOVA analysis with individual sam- isolation-by-distance in a one-dimensional distribution pling sites without any a priori pooling of populations

Fig. 2 Statistical parsimony haplotype network showing the phylogenetic relationship among 32 unique haplotypes observed among ten populations of M. simplex.Thecirclesarethehaplotypesandtheirsizerepresentstheirfrequencies in the total sample, small and white circles are missing estimated haplotypes. Each color corresponds to the populations given in Table 1 and Fig. 1 Cardoso et al. BMC Evolutionary Biology (2015) 15:106 Page 7 of 13

Fig. 3 (See legend on next page.) Cardoso et al. BMC Evolutionary Biology (2015) 15:106 Page 8 of 13

(See figure on previous page.) Fig. 3 Geographical distribution of all 32 cytochrome oxidase I (COI) unique haplotypes observed across the distribution of M. simplex along Atlantic Brazilian coast. The circles are the haplotypes and their size represents their frequencies in the total sample, singletons were suppressed and are shown in white. Colors display frequent haplotypes distributed throughout M. simplex distribution along Atlantic coast. The map was produced using a GIS program with free layers available at IBGE website (www.mapas.ibge.gov.br). The colors of haplotypes do not refer to the previous figures was not able to detect a best grouping scenario or pos- ago (Fig. 5b, dashed line). We could observe that the ex- sible breaks among sites (data not shown). pansion had a second increasing cline around 25,000 years ago, corroborating the observed mismatch distribution Demographic history (Fig. 5a). As a shallow phylogeographic structure was observed in M. simplex populations, we conducted a historical dem- Discussion ography analysis for the complete set of haplotypes. The The impact of Quaternary climatic fluctuations and geo- neutrality tests allowed identifying some statistical sig- logical events on the biodiversity of the Brazilian Atlan- nificant signatures for expanding population for both tic Forest has been extensively discussed based on tests. According to the extremely negative and signifi- studies on the genetic structure of forest dweller species. cant values of Tajima’s D (−1.47062, p = 0.0422) and Fu’s Phylogeographic studies of AF species often show a clear FS (−21.59803 p = 0.0001), haplotype frequencies differed division between northern and southern populations from those expected for a neutral population. Yet, the (e.g. [13, 14]), indicating that rivers may act as physical mismatch distribution of pairwise nucleotide differences barriers to gene flow [13] and that species were re- between haplotypes was bimodal (Fig. 5a) as expected stricted to fragmented forest refugia during glacial pe- for populations at demographic equilibrium. However, riods [10, 14, 15]. none of the models of population expansion could be Here, we contribute to this discussion by exploring the rejected. Harpending’s raggedness h statistic (R2) and the phylogeography of the ant M. simplex, which occurs in sum of square differences (SSD) supported a close fit to drier, sandy coastal plain habitats along the Atlantic the observed distribution under a pure demographic ex- coast in Southern Brazil. In contrast to the above- pansion model (SDD = 0.00754, p = 0.62760; R2 = 0.01864, mentioned studies on forest species (e.g. [14, 15]), our p = 0.86400) and under a sudden spatial expansion model data did not reveal a strong phylogeographic pattern (SSD = 0.00905, p = 0.40100; R2 = 0.01864, p = 0.84750). throughout the whole distribution of M. simplex. The The time to the most recent common ancestor (tMRCA) spatial analysis of population structure implemented by for all M. simplex haplotypes was estimated at 0.197 Mya SAMOVA did not suggest a congruent grouping, and we (with 95 % highest posterior density of 0.07-0.3559). The could not identify geographical barriers to gene flow. In analysis of the Bayesian Skyline Plot provided an add- contrast to other organisms, large bodies of water, such itional strong support for the evidence of past population as rivers or the estuarine complex of the Paranaguá Bay, ap- expansion (Fig. 5b). The results suggested that M. simplex parently do not impair the gene flow among the northern has undergone a long-term period of demographic popula- populations, and the populations between Florianópolis tion stability since the tMRCA lasting from ~ 70,000 years (SC) and “Ilha Comprida” (SP) were not genetically different.

Table 3 - ΦST values for pairwise comparisons between population of M. simplex (lower left) and p values (upper right) RS1 RS2 RS3 SC4 SC5 SC6 SC7 SC8 PR9 NE10 RS1 - 0.54618 0.01228 0.04633 0.18008 0.01792 0.01594 0.04841 0.08653 0.0492 RS2 0.01856* - 0.01683 0.00515 0.0302 0.02624 0.01465 0.04742 0.09554 0.0592 RS3 0.11411 0.13078 - 0.27888 0.02287 0.00386 0.00337 0.00485 0.01297 0.00941 SC4 0.09502 0.1619 0.01451** - 0.08455 0.00356 0.00297 0.00752 0.01733 0.01129 SC5 0.02839 0.093 0.10675** 0.07119** - 0.00069 0.0004 0.00168 0.00713 0.00614 SC6 0.27381 0.24143 0.34481 0.34975 0.30795 - 0.78299 0.92516 0.6338 0.83912 SC7 0.29673 0.27747 0.36158 0.35894 0.32158 0.05903*** - 0.59489 0.43421 0.47352 SC8 0.19818 0.17864 0.28682 0.26863 0.24282 0.06991*** 0.03956*** - 0.93931 0.77794 PR9 0.13621 0.12338 0.23448 0.21937 0.1883 0.06591*** 0.0298*** 0.08068*** - 0.94852 NE10 0.18098 0.16623 0.27856 0.27675 0.23074 0.07182*** 0.03535*** 0.06859*** 0.0855*** - Population names are given in the Table 1. The colors show the shallow phylogeographic structure found: southern populations (*), central-eastern population (**) and northern populations (***) Bold values are significant at P < 0.05 Cardoso et al. BMC Evolutionary Biology (2015) 15:106 Page 9 of 13

reflecting history. Besides, results can be affected by nat- ural selection [45]. Tropical forests (including the humid Atlantic Forest) were reduced during the Quaternary ice ages, imposing the contraction of species distribution and vicariant process [1, 6]. Yet, the drier and cooler environment during the ice ages promoted the expansion of open scrub environments in southern Brazil, including the sand dune areas along the Atlantic coast [20, 46]. Such dunes are the current habitat of M. simplex and the ab- sence of a strong phylogeographic structure is therefore in agreement with the geomorphologic history of this area, suggesting that M. simplex may not have experi- enced shifts in its distribution. This result is consistent with phylogeographic patterns reported for a coastal or- chid species with a similar distribution range [17]. To- gether, these results suggest that coastal lowland species may have had rather stable distributions during the past climatic oscillations. Similar patterns have been reported for other sand dune or coastal species worldwide [47–50]. Pairwise ΦST analysis allowed us to identify some re- strictions to gene flow among populations from the three major geographical regions (Table 3). This might be a result of some degree of isolation by distance among the populations of M. simplex. Indeed, genetic and geographic distances correlated weakly (see Results). Besides, more frequent haplotypes show a wide geo- graphic distribution (e.g., H3 and H11). This may reflect an ongoing dispersal along the coasts after LGM that continues until today. Alternatively, recurrent gene flow among nearby populations may have deleted a possible signal of the early history of this ant species. Pleistocene sea levels fluctuated considerably through the glacial and interglacial periods during the Quater- Fig. 4 Spatial analysis of molecular variance - SAMOVA of populations of M. simplex. a Fixation indices calculated and b percentage of genetic nary. During the Pleistocene, the coastal plain of the variation explained by each hierarchical level for the best grouping southern region of South America was larger than it is option for each pre-specified K groups now [51, 52]. Palaeo-geomorphological studies showed that the sea-level was about 120 m lower than present (Figs. 1 and 5b), leading to an elongation of the coast- line limits to ~100 km [20, 53]. The ocean regression re- In sum, our results suggest that populations of M. simplex sulted in the enlargement of the coastal lowland. The are not strongly isolated from one another and that gen- availability of new areas for M. simplex may have led to etic bottlenecks have been rare in the past. The phylogeo- the expansion of its populations. We found genetic sig- graphic pattern of M. simplex is probably explained best natures that supported this hypothesis. The negative and by an enduring persistence of its populations along the highly significant Fu’s FS and Tajimas’s D showed depar- seashore of the Atlantic coast and unrestricted gene flow tures from neutrality. Hence, the mismatch distribution along the coast. detected imprints of both sudden and spatial demo- However, our results should be viewed with some cau- graphic expansions in the past population of M. simplex. tion. As the examined nuclear markers were invariable The historical demographic reconstruction based on a and not informative our conclusions are based solely on Bayesian Skyline Plot showed that M. simplex underwent mitochondrial sequence data. Thus, such conclusions a stable demographic period shifting to a persistent might in general be biased to some extent due the mode growth of effective population size in recent periods. of inheritance, mutation rate and effective population Population growth started around 70,000 years ago and size of mitochondrial markers rather than accurately displays a slight increase around 25,000 years ago. These Cardoso et al. BMC Evolutionary Biology (2015) 15:106 Page 10 of 13

Fig. 5 Demographic history of M. simplex and relative sea level. a Pairwise mismatch distribution of the mtDNA sequences for total dataset. M. simplex presented a bimodal distribution, but did not reject the spatial expansion model (SDD = 0.0226 p=0.3803). b Bayesian skyline plot showing the historical demography and complete reconstruction of female effective population size fluctuations throughout the time of M. simplex. Black line represents median estimation and the grey area the upper and lower 95 % confident intervals. Dashed line indicates the beginning of demographic expansion. Blue line shows the sea-level during the last 150,000 years during the Quaternary (from [66]) results indicate that the processes underlying the current have been erased by population declines during the diversity of M. simplex occurred during the middle- demographic history of this ant [54]. It is possible that Pleistocene and intensified under different climatic and M. simplex populations underwent a demographic con- geological events imposed by the Last Glacial Maximum traction during LGM and then expanded thereafter, with (LGM), at the Late-Pleistocene. We cannot exclude, the contraction not being clearly evident in the BSP. however, other potential scenarios due the limitations of Nevertheless, historical expansions in the demography analyses based on a single locus. The historical scenario of M. simplex populations would be in agreement with of population growth of M. simplex starting during two events of lower historic sea-levels, i.e., the historic LGM could be a result of over-interpretation of BSP demographic expansion of M. simplex might have started [54], and signals about other population cycles could and persisted during the major period of sea-level fall (see Cardoso et al. BMC Evolutionary Biology (2015) 15:106 Page 11 of 13

Fig. 5b). Later, when the sea level rose to its actual status, mountainous relief of Serra do Mar [62]. As an effect of M. simplex populations could have persisted in areas re- Post Glacial Marine Transgression, the sea submerged lated to Pleistocene barriers. These barriers might have the majority of sandy beach ridges; removing M. simplex been sand deposits that were initiated to be accumulated from this portion of Brazilian coastline. They might have since the Last Interglacial around 120 Mya [2]. survived in the region of Cabo Frio because of the pres- The cooler climatic conditions during the Pleistocene ence of Pleistocene sand deposits that might have acted (lower around 3–7 °C less than today) may have not as refugia during the marine transgressive events [17]. been a constraint for the distribution of M. simplex, Cabo Frio and surrounding regions are the only area in since this species seems to be intolerant to warm tem- the south-eastern Brazilian coast with such aeolian dunes, peratures (Cardoso, pers. obs.). Today, M. simplex has a typical habitat of M. simplex [62]. an almost exclusively subtropical distribution, under the Furthermore, post-glacial AF expansion together with Tropic of Capricorn (23° S), except one residual popula- the rise of the sea level may have led to the shrinkage of tion at Cabo Frio beach in Rio de Janeiro state. The sand dune habitats between 23° and 24° S [63]. Finally, present distribution again suggests the important role of M. simplex co-occurs with M. conformis north of São ocean transgression. Its northernmost limit coincides Paulo State. Both species nest near the seashore and use with the major enlargement of the coastal lowland in debris from the sparse vegetation to grow their fungus Southern Brazil, from southern Chuí in Rio Grande do gardens (see [64] for nesting details). M. conformis seems Sul State to Cabo Frio in Rio de Janeiro State (Fig. 1) to tolerate a broader range of ecological conditions nest- [53] (see also [1]). The resulting new land bridges may ing in sunny and shaded environments of sand dunes have enabled migration of many organisms across the and the workers are active during the day and night latitudinal gradient by forming sand corridors or sand (Cardoso, pers. obs.). Yet, M. simplex exclusively nests inlands due to coastal deposits. in sunny sand dune places and its workers are active Bodies of water resulting from the regression and only during dusk and overnight [65]. All three factors transgression of the sea apparently did not present an may have reduced the local density of M. simplex and fi- obstacle for flying and did not impair the disper- nally led to its extinction in the area between southern sion of ants [55]. Sandy beach corridors and islands were São Paulo and Cabo Frio. Indeed, M. conformis was formed during at least three phases, with sea levels simi- found living in the northern range where M. simplex lar to the current level until 6,500 years ago [53]. Thus, was not found, beside our sampling effort. M. simplex may have had enough time to expand its range in an almost panmictic population. Sexuals of Conclusions fungus-growing ants typically mate during nuptial flights. Phylogeographic studies regarding species associated with Winged male and female sexuals leave their natal colonies. open and dry environments have attracted much less at- After mating, young queens then disperse downwind on tention compared to the number of studies using organ- the ground to establish a new colony [56]. The dynamics isms associated with forests as a study model. Identifying and timing of nuptial flights and subsequent dispersal dif- explicit phylogeographical patterns and the factors under- fer among species, but in general, the mating biology of pinning genetic structure are reasonably difficult, particu- ants remains remarkably unstudied [57, 58]. Leaf-cutter larly for those species inhabiting historic and dynamic ants with large queens, such as Atta and Acromyrmex,ap- regions, such as coastal sand dunes. Our findings indicate pear to have low dispersal distances [59, 60]. In contrast, that M. simplex presents an evolutionary history consistent queens with small body size, such as those of M. simplex, with shifts in the sea-level and changes in the distribution can stay in the air for considerable time and might fly for of dry vegetation in southeastern Brazil. The diversification kilometers [61]. In this way, M. simplex might be able to and expansion started in the mid-Pleistocene, during maintain gene flow and dispersal along the coast. which major climatic changes occurred worldwide. Our The absence of M. simplex between southern São results are in agreement with other studies with sand dune Paulo and Cabo Frio could be explained by three non- species that indicate expansion during glacial periods, but exclusive hypotheses: (i) Holocene marine transgression is contrasting with others, suggesting that a single and drowned the suitable habits in that M. simplex occurred; wide model of Quaternary effects on the diversification (ii) such open habitats were removed by the expansion and distribution of species is unrealistic. of the forests towards the seashore, which is currently covered by the tropical Atlantic rain Forest; (iii) compe- Availability of supporting data tition with congeneric species. All datasets supporting of this article are provided with Overall, Rio de Janeiro is characterized by a rocky Additional files. All gene sequences obtained in this coastline with little development of transitional sedi- study were deposited in the GenBank database under mentary coastal plains, due to the proximity of the the following accession numbers: wingless (KP939178- Cardoso et al. BMC Evolutionary Biology (2015) 15:106 Page 12 of 13

KP939228) and cytochrome oxidase subunit I (KJ842219- Received: 12 June 2014 Accepted: 20 May 2015 KJ8423260).

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Barker BS, Rodríguez-Robles JA, Aran VS, Montoya A, Waide RB, Cook JA. Sea variants. level, topography and island diversity: phylogeography of the Puerto Rican Additional file 2: Appendix S2. Bayesian phylogenetic consensus tree red-eyed Coquí, Eleutherodactylus antillensis. Mol Ecol. 2012;21:6033–52. of COI gene sequences of M. simplex haplotypes and outgroups. The 5. Stewart JR, Lister AM. Cryptic northern refugia and the origins of the numbers at branches are Bayesian posterior probabilities (P.P.). Tree modern biota. Trends Ecol Evol. 2001;16:608–13. branches are proportional to mutational events sampled in sequences 6. Hewitt GM. The structure of biodiversity - insights from molecular alignment (scale bar). phylogeography. Frontiers in zoology. 2004;1:4. 7. Hewitt G. Quaternary phylogeography: the roots of hybrid zones. Genetica. 2011;139:617–38. Abbreviations 8. Vialatte A, Guiller A, Bellido A, Madec L. Phylogeography and historical AF: Brazilian Atlantic Forest; AIC: Akaike’s Information Criterion; BSP: Bayesian demography of the Lusitanian snail Elona quimperiana reveal survival in skyline plot; COI: Cytochrome oxidase subunit I; CTAB: Cetyltrimethyl unexpected separate glacial refugia. BMC Evolutionary Biology C7 - 339. ammonium bromide; DNA: Deoxyribonucleic acid; dNTPs: Deoxynucleotides; 2008;8:1–13. ESS: Effective sample sizes; Γ: Gamma distribution; GIS: Geographic 9. Widmer I, Dal Grande F, Excoffier L, Holderegger R, Keller C, Mikryukov VS, information system; GTR: General time reversible model; I: Proportion of et al. European phylogeography of the epiphytic lichen fungus Lobaria invariable sites; IBGE: Brazilian Institute of Geography and Statistics; LGM: Last pulmonaria and its green algal symbiont. Mol Ecol. 2012;21:5827–44. Glacial Maximum; MCMC: Markov Chain Monte Carlo; mtDNA: Mitochondrial 10. Carnaval AC, Hickerson MJ, Haddad CFB, Rodrigues MT, Moritz C. Stability DNA; Mya: Million years ago; PCR: Polymerase Chain Reaction; PP: Posterior predicts genetic diversity in the Brazilian Atlantic Forest hotspot. Science. probability; SAMOVA: Spatial analysis of molecular variance; SD: Standard 2009;323:785–9. deviation; SSD: Sum of square differences; tMRCA: The most recent common 11. Carnaval AC, Moritz C. Historical climate modelling predicts patterns of ancestor. current biodiversity in the Brazilian Atlantic forest. J Biogeogr. 2008;35:1187–201. 12. Myers N, Mittermeier RA, Mittermeier CG, da Fonseca GAB, Kent J. Competing interests Biodiversity hotspots for conservation priorities. Nature. 2000;403:853–8. The authors declare that they have no competing interests. 13. Grazziotin FG, Monzel M, Echeverrigaray S, Bonatto SL. Phylogeography of the Bothrops jararaca complex (Serpentes: Viperidae): past fragmentation and Authors’ contributions island colonization in the Brazilian Atlantic Forest. Mol Ecol. 2006;15:3969–82. The concept of this study was developed by DCC and MPC. Field and 14. Fitzpatrick SW, Brasileiro CA, Haddad CFB, Zamudio KR. Geographical laboratory work was conducted by DCC and MPC; DCC, MPC, CDS, JH carried variation in genetic structure of an Atlantic Coastal Forest frog reveals out the analysis of the data and drafted the manuscript; all authors read, regional differences in habitat stability. Mol Ecol. 2009;18:2877–96. discussed and approved the final version of the paper. 15. Thomé MTC, Zamudio KR, Giovanelli JGR, Haddad CFB, Baldissera Jr FA, Alexandrino J. Phylogeography of endemic toads and post-Pliocene persistence Acknowledgments of the Brazilian Atlantic Forest. Mol Phylogenet Evol. 2010;55:1018–31. The present work could not be accomplished without help of many people. 16. Resende HC, Yotoko KSC, Delabie JHC, Costa MA, Campiolo S, Tavares MG, I would like to thanks Lucinda Lawson for helping us during BEAST analysis et al. Pliocene and pleistocene events shaping the genetic diversity within and Nicole Rivera in the course of population genetics analysis. We are the central corridor of the Brazilian Atlantic Forest. Biol J Linn Soc. grateful to Vivian Sandoval Gomez and Fátima Maria dos Passos Cristiano for 2010;101:949–60. their help in sampling field. We are also thankful Andreas Trindl and Doris 17. Pinheiro F, de Barros F, Palma-Silva C, Fay MF, Lexer C, Cozzolino S. Rothgänger for their assistance in laboratory works. This research was Phylogeography and genetic differentiation along the distributional range majorly supported by Fundação de Amparo à Pesquisa do Estado de Minas of the orchid Epidendrum fulgens: a neotropical coastal species not restricted Gerais – FAPEMIG (Process number: CRA-APQ-00540-11), and additional to glacial refugia. J Biogeogr. 2011;38:1923–35. financial support was provided by Coordenação de Aperfeiçoamento de 18. Ribeiro RA, Lemos-Filho JP, Ramos ACS, Lovato MB. 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